The present invention is related generally to a keratin composition and method for making same. Specifically, the present invention relates to an absorbent keratin powder or fiber. More specifically, the present invention includes a hydratable keratin solid which forms a hydrogel upon addition of water for use in various applications including nonwoven films, diapers, skin treatments, prosthetic devices, excipients, tissue engineering scaffolds and the like. Active agents can be bound to the keratin excipient for controlled drug delivery.
Absorbent materials are currently used to absorb body fluids such as urine, menses, and wound exudate. The absorbent materials are placed near the skin to serve this purpose. One class of products includes diapers, where the absorbent material can be derived from wood pulp, cellulosic fibers, or super absorbent, synthetically produced material. Diapers commonly have an inner core designed to absorb urine and water. The core is typically formed from a superabsorbent polymer dispersed in a larger amount of less absorbent material. The absorbent materials typically contained in the core are separated from the skin by at least one layer of material. The absorbent materials absorb urine and can become saturated. It is believed that some material from the absorbent core leaches from the wet absorbent and travels back to the skin. In the case of chemically treated absorbent materials and films, depending on the chemicals, the leachate may be irritating and is not believed to be beneficial. Skin contact with urine can also occur and result in irritation. This type of irritation may exacerbate diaper rash problems.
Other products which contain absorbent materials for use next to the skin include feminine hygiene products such as tampons and pads. These products serve to absorb menses. Another class of products using absorbent materials includes wound dressings, both those designed for humans, and dressings for veterinary use for application to wounds or skin irritations or disorders in animals. For specific applications, wound dressings preferably absorb exudate from wounds while keeping the wounds relatively moist to promote healing. In some applications, a gel may be desirable as a wound dressing, where the gel can maintain a moist wound environment, while absorbing excess exudate.
What would be desirable is an absorbent material formed from a natural product. What would be beneficial is a non-toxic product derived from natural sources that would cause no concern when leachate from the material contacts the body or the material itself contacts the body. What would be advantageous is a material that can absorb urine and, when wet, leach out a natural product that is beneficial with respect to diaper rash. What would be desirable is a material that can return a skin healing leachate to the skin. What would be desirable is a material that aids wound healing. What would be desirable is a hydrogel made of natural products formable by adding water to a powder or fiber. What would be desirable is a biocompatible carrier or excipient that could be used in the delivery of drug actives to various organs of the body. What would be desirable is a biocompatible carrier to which active agents can be bound and later released.
Tissue engineering is a rapidly growing field encompassing a number of technologies aimed at replacing or restoring tissue and organ function. The success of tissue-engineered implants rest on the invention of biocompatible materials that can act as cell-scaffolds and support cell growth. Of benefit are tissue-engineering scaffolds materials that are mitogenic or contain mitogenic factors. Such scaffolding materials can be used for in a wide array of tissue engineering implants containing cellular components, such as, for example, osteoblasts, chondrocytes, keratinocytes, and hepatocytes, to restore or replace bone cartilage, skin and liver tissue respectively.
The present disclosure addresses at least some of the deficiencies in the art by providing a hydratable, hydrogel-forming solid derived from a keratinous source such as hair, fur, human hair and the like. In the context of the present invention, the term xe2x80x9chydratable keratinxe2x80x9d and xe2x80x9chydratable keratin materialxe2x80x9d is a keratin or keratin material that when hydrated can form a hydrogel. In the context of the present invention, the term xe2x80x9ccross-linked insoluble oxidized keratinxe2x80x9d means a network of cross-linked keratin containing sulfonic acid residues that is effectively of infinite molecular weight in that the molecular weight is approximated by the weight of the sample. Such networks may be viewed as being effectively one molecule. The status of a sample as being a xe2x80x9ccross linked insoluble oxidized keratinxe2x80x9d can be verified by the addition of a solvent in which an oxidized keratin monomer (prepared by breaking all cross-links) is soluble. A xe2x80x9ccross-linked insoluble oxidized keratinxe2x80x9d will swell upon addition of such a solvent, but will not enter solution despite agitation, heat and prolonged incubation unless and until the cross-linked insoluble oxidized keratin undergoes degradation to form lower molecular molecules. The cross-linked insoluble oxidized keratin preparation of the current invention may contain up to about 90 percent by weight of lower molecular weight polypeptides which may be soluble, but the insoluble character and gel forming are dependent upon network structure of the cross-linked insoluble oxidized keratin. The term xe2x80x9cinsolublexe2x80x9d refers to the properties of the of cross-linked oxidized keratin while it exists as a network of effectively infinite molecular weight. Upon hydrolysis, the cross-linked insoluble oxidized keratin network will enter into solution to the extent of degradation to lower molecular weight polypeptides. As this process ensues, molecules of finite molecular weight are generated which continue to hydrolyze and degrade to molecules of decreasing molecular weight. The solubility of these lower molecular weight polypeptides is enhanced by the fact that hydrolysis results in the generation of molecules that are more soluble by virtue of their low molecular weight (Flory, 1953). Solubility is further enhanced by the increasing relative molar percentage of amine and acid functional groups that are being generated as a result of the continued hydrolytic degradation. The rate of such degradation can be controlled by varying preparation parameters of the xe2x80x9ccross-linked insoluble oxidized keratinxe2x80x9d such as extent of oxidation and the solids content. When the solvent is water, a cross-linked insoluble oxidized keratin that has undergone the ion exchange process of the current invention will form a hydrogel.
In certain embodiments, a hydrogel-forming solid as disclosed herein may absorb up to 5 to 20 times its weight in water to form a hydrogel. Such a solid, as well as the hydrogel formed from the solid will be useful in various applications such as use as an absorbent with skin healing properties when incorporated into diapers, feminine hygiene products, wound dressings, including both human and veterinary uses, as a soft tissue augmentation medium when used in subdermal implants, tissue engineering cell scaffolds, as a moisture containing agent in cosmetics, oils, lotions, or gels for use on the skin, in applications related to the healing of damaged skin, and as a pharmaceutical excipient for sustained and/or controlled release pharmaceutical applications.
A hydratable keratin solid may be made by methods that include providing a keratinous material, or keratin, having disulfide linkages and partially or substantially oxidizing the keratinous material with an oxidizing agent, for example, such that some disulfide linkages are cleaved and oxidized, forming hydrophilic sulfonic acid or cysteic acid residues. A preferred source of keratinous material is human hair, although the keratin may be obtained from hair or fur of animals including any mammal, or from finger or toenail material or from hooves, feet, beaks, skin, feather or horns. Human hair is a preferred source of keratin because of its ready availability from cuttings of barber and beauty shops, because it is expected to be less prone to cause undesirable immune or allergic reactions in a human, and because a keratin preparation may be made from the hair of a subject for whom the preparation will be used. This last advantage can be especially important in embodiments involving subdermal and tissue engineering implantations.
It is well known in the art that keratins contain substantial sulfur, that is, the amino acid sequence of keratin contains a high proportion of cysteine residues as compared to proteins in general. These cysteines each include a sulfhydryl moiety that is able to bond with another sulfhydryl moiety from another cysteine residue to form a disulfide bond. The second cysteine may reside within the same keratin molecule, or in another keratin molecule. These disulfide bonds are responsible for much of the tertiary and/or quaternary structure of this class of proteins. A suitable oxidizing agent is able to break this disulfide bond and to oxidize one or both of the sulfhydryl moieties so that they are no longer able to form a disulfide. Such an oxidation is a part of the process of forming the keratin products of the present disclosure. Preferred oxidizing agents include, but are not limited to peracetic acid, hydrogen peroxide, perborates, percarbonates, benzoyl peroxide, or ammonium sulfate peroxide. However, any suitable oxidizing agent known in the art can be used in the practice of the invention. After oxidation, the liquid oxidizing agent can be filtered from the oxidized keratin solid, and the solid may be washed to remove residual oxidizing agent, for example.
The resulting solid may then be suspended in a non-aqueous solvent and the pH may be adjusted upward with basexe2x80x94conveniently to at least neutral pH. Preferred solvents for this second solution do not include more than about 20 volume percent water, as the water may hydrolyze the peptide backbone during processing. Preferred solvents would include alcohols such as methanol, ethanol, or propanol, for example, and would also include non-aqueous polar, water-miscible solvents such as acetone and tetrahydrofuran, for example. An effective solvent should be able to solvate a Lewis base and should also be able to provide a medium able to keep the keratin sufficiently swelled to allow ionic associations or interactions between the base cations and anionic sulfonic acid groups in the keratin. Small amounts of water will assist in this regard, so blends of the aforementioned solvents in combination with water up to 20 volume percent may be used. Preferred bases include, but are not limited to sodium hydroxide, potassium hydroxide and ammonium hydroxide, which, as is known in the art, would yield or produce sodium, potassium and ammonium cations, respectively, upon entering solution.
The keratin suspension may be heated, and is preferably heated to boiling for a time sufficient to swell the keratin. The keratin suspension may be stirred without heat for a longer period of time to allow a more complete association or reaction between the sulfonic acid groups and the base cations. The continued reaction time at or near room temperature, or even below room temperature while stirring is contemplated by the inventors to allow the base cations to approach and bind to the keratin anionic sites with a lower incidence of peptide backbone degradation that could occur with continued boiling. The cations for use in the present invention, therefore, must be able to interact with the anionic cysteic groups in the keratin material. The use of the term xe2x80x9ccationsxe2x80x9d or xe2x80x9cmonovalent cationsxe2x80x9d in the present disclosure and claims is indication of those cations that are able to do so. Salts of aspartate and glutamate may also be present in high concentration and will contribute to the absorbency of the hydratable keratin material. After a sufficient reaction time, the keratin solid may be removed from the suspension by filtration, for example, and dried, leaving a solid salt formed of the keratin sulfonic acid or cysteic acid groups and base cations. This solid may be shredded into a fibrous form and/or ground into a finely divided powder. This solid may be used in certain embodiments, or it may be hydrated by adding water, for example, and the hydrogel, or viscoelastic hydrogel thus formed may be used in certain embodiments.
In certain embodiments, an absorbent keratin layer may be incorporated into various absorbent articles such as a disposable diaper, a wound dressing, or feminine hygiene product, by adsorbing or coating a keratin solid or hydrogel onto a layer of the article, by impregnating a component of such an article, or by associating a keratin material with a nonwoven layer of such an article. In certain embodiments an absorbent keratin powder may be applied directly to skin to absorb moisture and inhibit rashes or chafing, such as diaper rash, for example. A hydratable keratin solid of the invention may have an absorbency of 1, 5, 10, 15 or even up to 20 times its weight in water. The absorbency may be adjusted by, for example, varying the degree of oxidation of the keratin in the process. It may thus provide a substitute or a supplement for products such as talc and cornstarch. The inventors have demonstrated, for example, that a fibrous or powdered form of solid keratin material as described herein can absorb about 10 times its weight in water in about 10 seconds.
The hydratable keratin solids as described herein form a hydrogel or a viscoelastic hydrogel upon application of water, and also are contemplated to contain skin healing peptides associated with the keratin, which may leach out of the keratin products when wet. The keratin products thus provide an added benefit, in addition to water absorbency, that is, healing or soothing peptides are also released that may have beneficial effects on the skin of a user of the products. This property offers certain benefits in embodiments such as wound dressings, as well as cosmetics, gels or lotions for application to the skin.
In certain embodiments a keratin absorbent as disclosed herein may be used as a wound dressing material to absorb wound exudate by direct application, or by incorporation into a dressing. The solid, hydratable forms of keratin offer certain advantages in such applications because they may be stored as dry powders or fibers and hydrated to form a gel in the field, or only as needed, for example. Medical applications, such as wound exudate management or drug release, can make use of the keratin material in absorbent powder, fiber, woven fiber, or felt form.
The keratin hydrogel is also believed to be suitable for use as an implant filler, for example, used to fill a breast implant, or to augment soft tissue for cosmetic, reconstructive or aesthetic reasons, or in a tissue expander application. The keratin product may also be used in cosmetics to retain moisture next to the skin. The performance of cosmetics which reduce the greasy appearance of skin can be enhanced through the use of moisture absorbent keratin material as an additive or base ingredient, for example, in a cosmetic formulation. The keratin absorbent and hydrogel can also be used for a variety of tissue engineering applications. Both materials may act as biocompatible scaffolds that provide a mitogen, the keratin peptide, to the cellular components of a tissue-engineered implant. In the case of a keratin hydrogel tissue engineered implant, the degradation of keratin to lower molecular weight peptides can be controlled through a combination of processing and formulation parameters. As with other materials known in the art, the degradation rate is directly related to the rate of resorbtion in-vivo (Agrawal, 1997). Therefore, the resorbtion rate of the keratin hydrogel can be directly controlled.
The present invention may be described, therefore, in certain aspects as a composition comprising a hydratable keratin solid, wherein the solid comprises a keratin where at least a portion of the cysteic groups of the keratin are ionically or electrostatically associated with, or may be ionically bound to cations. As used herein, ionically bound or ionically associated would have their ordinary meaning as is known in the art, and would include the electrostatic attraction between an anion and a cation, and would include such interactions directly, such as through formation of ionic bonds, and interactions through intermediary bipolar moieties, for example. A cysteic group would include cysteine and derivatives of cysteine including cysteine and cysteic acid or sulfonic acid. As used herein, cysteic acid and sulfonic acid denote a cysteine side chain in which the terminal sulfur is bonded to three oxygen atoms to produce the sulfonic acid ion, SO3xe2x88x92, or the acidic form, SO3H. In certain embodiments, a portion of the cysteic groups are oxidized to sulfonic acid or cysteic acid groups. Sulfonic acid or cysteic acid groups may comprise a significant portion of the total cysteic groups and in some embodiments the sulfonic acid groups may constitute a major portion of the total cysteic groups. The extent of the oxidation may be adjusted by adjusting certain parameters of the oxidation reactions, such as temperature, concentration of oxidizing agent, and time of reaction, for example, to achieve a product with certain desired properties, such as absorbency or resiliency, for example.
In certain embodiments, therefore, the present invention may be described as a hydratable keratin solid made by a process comprising oxidizing a portion of the cysteic acid groups of a keratin to obtain a keratin having oxidized cysteic groups, and contacting the keratin having oxidized cysteic groups with monovalent cations under conditions effective to form ionic associations or ionic bonds between at least a portion of the oxidized cysteic groups and the cations.
In some embodiments, the hydratable keratin solid is made by a process comprising oxidizing at least a portion of the cysteic acid groups of a keratin to obtain a keratin having oxidized cysteic groups, and contacting said keratin having oxidized cysteic groups with monovalent cations under conditions effective to form ionic associations or ionic bonds between a substantial portion of said oxidized cysteic groups and said cations. The oxidation may comprise placing the keratin in a solution containing a concentration of an oxidizing agent effective to oxidize a portion of the cysteic acid groups. The portion of oxidized cysteic groups may be a major portion of the total cysteic acid groups.
In certain embodiments of the present invention, the oxidation comprises placing the keratin in a solution containing a concentration of hydrogen peroxide, peracetic acid, perborates, percarbonates, benzoyl peroxide or ammonium sulfate peroxide effective to oxidize a portion of the cysteic groups.
The process of the present invention may further comprise heating the keratin solid containing oxidized cysteic groups in a solvent solution containing a dissolved base, wherein the base produces the monovalent cations in the solution. The solvent solution may comprise a solvent selected from methanol, ethanol, propanol, ether, tetrahydrofuran (THF), acetone, propylene glycol, 1,4-dioxane, and glycol ether, or combinations of these with up to 20 volume percent water. In certain embodiments the process further comprises removing the solution from the heat and stirring for a time effective to form ionic bonds between the cysteic groups and cations produced by the base. The process may also further comprise drying the keratin solid, such as by drying a solid or solution under vacuum.
Another aspect of the present invention is a composition comprising a keratin hydrogel wherein the hydrogel is produced by adding water to a composition comprising a hydratable keratin solid, wherein the solid comprises a keratin where at least a portion of the cysteic groups of the keratin are ionically bound to cations. In some embodiments, the composition of the present invention comprises a keratin viscoelastic hydrogel produced by adding water to a composition comprising a hydratable keratin solid, wherein the solid comprises a keratin where a portion of the cysteic groups of the keratin are ionically bound to or associated with cations.
Another aspect of the present invention is a process for making a hydratable keratin solid comprising: (1) oxidizing keratin in a first solution comprising a soluble oxidizing agent, such that a portion of the disulfide bonds of the keratin are oxidized to form sulfonic acid residues, to obtain an oxidized solid fraction; (2) separating the oxidized solid fraction from the first solution; (3) contacting the oxidized solid fraction with a second, basic solution comprising a monovalent cation dissolved in a solvent or solvent mixture; (4) maintaining the second solution containing the oxidized solid fraction and the monovalent cations for a time and at a temperature effective to allow an interaction between the sulfonic acid residues and the monovalent cations to obtain a salt solution of the keratin and the monovalent cation; and (5) substantially removing the solvent from the salt solution to obtain a pure hydratable keratin solid.
The process may also further comprise adjusting the pH of the second solution, to obtain a substantially neutral solution. In some embodiments, the keratin is obtained from hair, fur, skin, feet, beaks, horns, hooves or feathers and is preferably obtained from human hair.
In some embodiments, the keratin is oxidized by suspending the keratin in a solution of a suitable oxidizing agent such as one selected from the group consisting of hydrogen peroxide, peracetic acid, perborates, percarbonates, benzoyl peroxide, and ammonium sulfate peroxide, in a concentration of between about 1 and about 35 weight/volume percent. In various embodiments, the keratin is oxidized by suspending the keratin in a solution of an oxidizing agent selected from the group consisting of hydrogen peroxide, peracetic acid, perborates, percarbonates, benzoyl peroxide, and ammonium sulfate peroxide, in a concentration of about 1, or about 2, or about 3, or about 4, or about 10, or about 15, or about 20, or about 30, or about 32, or about 35 weight/volume percent. As used herein the term weight/volume percent refers to a solution in which the concentration is determined in weight percent, that is then diluted into a particular volume, arriving at a weight/volume percent. For example, in order to arrive at the oxidant solutions described herein a xe2x80x9cstock solutionxe2x80x9d at fairly high concentration is diluted in water. As an example, hydrogen peroxide may be purchased as a 30 weight % solution (30 grams of peroxide per 100 grams of solution). To make 1 liter of a 2% solution of this, one would dilute 66.7 mL of the 30 weight % stock solution in 933.3 mL of water. The net effect is to cut the stock solution 15-fold (from 30 down to 2%). This ratio is a weight to volume ratio, so the resulting solution is described as 2 weight/volume %.
In some embodiments, the keratin is oxidized by suspending the keratin in a solution of a suitable oxidizing agent, such as one selected from the group consisting of hydrogen peroxide, peracetic acid, perborates, percarbonates, benzoyl peroxide, and ammonium sulfate peroxide, in a concentration of between about 1 and about 35 weight/volume percent, at a temperature between about 0xc2x0 C. and about 100xc2x0 C. In other embodiments the temperature is between about 4xc2x0 C. and about 90xc2x0 C., or between about 20xc2x0 C. and about 100xc2x0 C., or between about 80xc2x0 C. and about 100xc2x0 C. In other embodiments, the temperature is about 4xc2x0 C., or about 90xc2x0 C., or about 100xc2x0 C.
The present invention may also include the process wherein the keratin is oxidized by suspending said keratin in a solution of an oxidizing agent selected from the group consisting of hydrogen peroxide, peracetic acid, perborates, percarbonates, benzoyl peroxide, and ammonium sulfate peroxide, in a concentration of between about 1 and about 35 weight/volume percent, at a temperature between about 0xc2x0 C. and about 100xc2x0 C. for a period of between 0.5 and about 24 hours, or in a concentration of oxidizing agent of between about 1 and about 35 weight/volume percent, at a temperature between about 0xc2x0 C. and about 100xc2x0 C. for a period of between 1 and about 2 hours, or for between about 2 and about 4 hours, or for between about 1 and about 4 hours, or for a period of about 10 hours.
More specifically, the present invention may include oxidizing the keratin by suspending the keratin in a solution of between about 1 percent to about 32 percent peracetic acid at a temperature between about 0xc2x0 C. and about 100xc2x0 C. for between about 0.5 and about 24 hours, or by suspending the keratin in a solution of about 1 percent peracetic acid at a temperature between about 0xc2x0 C. and about 100xc2x0 C. for between about 0.5 and about 24 hours, or by suspending the keratin in a solution of between about 4 percent peracetic acid at a temperature of about 4xc2x0 C. for 24 hours, or by suspending the keratin in a solution of about 4 percent peracetic acid at room temperature for about 24 hours, or by suspending the keratin in a solution of about 4 percent peracetic acid at about 90xc2x0 C. for about 10 hours, or by suspending the keratin in a solution of about 4 percent peracetic acid at a temperature between about 20xc2x0 C. and about 100xc2x0 C. for between about 1 and about 4 hours, or by suspending the keratin in a solution of about 4 percent peracetic acid at a temperature between about 80xc2x0 C. and about 100xc2x0 C. for between about 1 and about 2 hours, or even by suspending the keratin in a solution of about 2 percent peracetic acid at a temperature between about 0xc2x0 C. and about 100xc2x0 C. for about 2 hours.
A second solution in the process of making the disclosed compositions, wherein the second solution contains the oxidized solid fraction and monovalent cations may be heated, and may also be boiled for between about 0.5 hours and about 12 hours, for between about 0.5 hours and about 3 hours, or for about 1 hour. Once said solution is boiled, the solution may be allowed to continue reacting while being stirred after removal of the heat. Alternatively, the solution may be stirred and allowed to react without the application of heat, or of boiling temperatures. In certain embodiments, the solution is allowed to react at a temperature of between about 15xc2x0 C. and about 30xc2x0 C. for a period of between about 1 and about 24 hours, or at a temperature of between about 20xc2x0 C. and about 25xc2x0 C. for a period of between about 1 and about 5 hours, or at room temperature for a period of about 5 hours. In certain preferred embodiments the solution is heated to the boiling point of the solvent and boiled for 2 hours.
Certain processes as described herein are effective to produce a hydratable keratin solid, and it is an embodiment of the present invention that those solids may be hydrated by the addition of water to obtain keratin hydrogels, or even viscoelastic keratin hydrogels. The terms hydrogel and viscoelastic hydrogel, as used herein, are meant to have the art recognized definition, and could be described as absorbing water such that the water cannot be removed by mechanical methods such as pressure or centrifugation. Viscoelastic hydrogels would also be defined as gels that display non-Newtonian fluid properties.
In certain embodiments the present invention may be described as a disposable diaper that includes a hydratable keratin solid, or a diaper which incorporates a hydratable or absorbent keratin solid. A hydratable keratin solid may be coated on a layer of the diaper, either a layer next to the skin of a wearer, or a layer separated from the skin of a wearer by a water permeable layer. In certain embodiments a hydratable keratin solid may be associated with a nonwoven layer of a diaper, or may be impregnated into a layer of a disposable diaper, or it may be contained in an inner absorbent core.
In certain alternative embodiments, the present invention may be described as a feminine hygiene product, or a wound dressing that includes a hydratable keratin solid. As was described for use in diapers, a hydratable keratin may be coated on a layer of a product, associated with a nonwoven layer of a product, or even impregnated into a layer of a product or contained in an absorbent core. Exemplary products would include wound dressings, tampons, and sanitary pads. Wound dressings include absorbent wound dressing, that is dressing capable of, but not limited to, absorbing wound exudate and blood. Absorbent dressings include, but are not limited to, adhesive bandages. Adhesive bandages typically comprise an absorbent pad, a backing and a pressure sensitive adhesive to maintain the dressing in place. In one aspect of the present invention, an absorbent wound dressing comprising hydratable keratin is an absorbent pad of an adhesive bandage. In one embodiment, the hydratable keratin in such an absorbent pad is in a nonwoven film.
Certain embodiments of the invention may be described as methods for promoting healing of skin in a subject including a human or an animal having damaged skin, including providing an absorbent, keratin material, wherein a portion and preferably a substantial or major portion of the cysteic groups of said keratin are oxidized and wherein water soluble peptides are associated with the keratin, wherein at least some of said peptides can leach out from said keratin upon application of water, and wherein said peptides promote healing of damaged skin; and disposing the absorbent keratin material near damaged skin, such that moisture causes at least some of said peptides to leach out of said keratin and to contact said skin. The method may be practiced with animal or human subjects, such that either animal or human skin is healed by this method. The practice of the method for promoting skin healing as described herein may include the treatment of damaged skin including, but not limited to diaper rash, burn, sunburn, cut, abrasion, puncture, a sore, bed sore, ulcer, diabetic ulcer, irritated skin, surgical incision, skin graft donor site, or wrinkled skin. The keratin material may be incorporated in a nonwoven film. The nonwoven film may comprise synthetic polymer webs and may also comprise natural materials such as cotton. It is understood that in the practice of such embodiments, the wound of the subject being treated may exude or excrete moisture and that the absorption of such moisture by said keratin may cause the release of water soluble peptides from keratin products of the present invention.
In certain embodiments the present invention may be described as a method for promoting skin healing, in particular in those embodiments in which a keratin solid or hydrogel as described herein, such as a keratin solid or hydrogel in which the keratin is obtained from human hair, for example, is contained in, or forms a portion of a cream, lotion, or gel for application to skin, hair, lips, or nails, for example. Such formulations can offer various advantages such as moisturizing the skin, or inhibiting loss of moisture from the skin, as well as providing the healing effects of peptides that may leach from the keratin containing product. Such creams, lotions and gels may be applied to damaged skin, such as dry, burned, sunburned, wrinkled, cut, scraped, chapped, irritated, ulcerated or otherwise damaged skin or other tissue.
One aspect of the present invention is a nonwoven film composition comprising a synthetic polymer and a keratin material, wherein the keratin material has been oxidized and contains sulfonic acid groups. The keratin material may be oxidized keratin material that has not undergone the ion exchange process or may be oxidized keratin that has undergone the ion exchange process to form a hydratable keratin. In the latter, the sulfonic acid groups of the hydratable keratin are associated with monovalent cations. The keratin materials may also be associated with pharmaceutical agents which may be in the form of polar compounds which are capable of binding or otherwise associating with the keratin. Such a pharmaceutical agent is asprin. The synthetic polymer may be, but is not limited to, xcex1-olefins, acrylates, urethanes, acetates, nylons, esters, and copolymers thereof. An xcex1-olefin is considered to be any monomer containing an xcex1-double bond. The nonwoven composition may also further comprise a natural material which may be, but is not limited to, cotton. In some embodiments of the invention, the nonwoven composition is a laminate, which may be, but is not limited to, a tri-laminate comprising two outer layers of synthetic polymer and a middle layer of keratin material. The keratin material in the middle layer may be partially exposed by openings in the two outer nonwoven synthetic polymer layers. In some embodiments of the invention the synthetic polymer layers are nonwoven webs of polymer fibers. In other embodiments, the synthetic polymer layers are woven webs of polymer fibers.
Another aspect of the invention is a nonwoven tri-laminate composition comprising a middle layer of a keratin material between two outer layers of synthetic polymer material. The synthetic polymer may be in the form of a nonwoven web. The keratin material may be oxidized keratin that contains sulfonic acid residues. The oxidized keratin may be subjected to ion exchange such that the keratin material is a hydratable keratin material. The keratin material may be associated with pharmaceutical agents, which may be in a cationic form. The synthetic polymer may be, but is not limited to, xcex1-olefins, acrylates, urethanes, acetates, nylons, esters, and copolymers thereof. The synthetic polymer may be, but is not limited to, poly(hydroxy acids) such as polylactic acid, polyglycolic acid, or a copolymer thereof. The nonwoven composition may also further comprise a natural material which may be, but is not limited to cotton.
One aspect of the invention is a process for making a nonwoven film. In one embodiment a keratin material is applied to a first nonwoven web layer of synthetic polymer. A second nonwoven web layer of synthetic polymer is applied over the keratin material so as to form a tri-laminate composition with two outer layers of nonwoven synthetic polymer web and a middle layer of keratin material. Another aspect of the invention is a product made by the above described process. The keratin material may be oxidized keratin that contains sulfonic acid residues. The oxidized keratin may be subjected to ion exchange such that the keratin material is a hydratable keratin material. The keratin material may be associated with pharmaceutical agents, which may be in a cationic form. The synthetic polymer may be, but is not limited to, xcex1-olefins, acrylates, urethanes, acetates, nylons, esters, and copolymers thereof. The nonwoven composition may also further comprise a natural material which may be cotton. The keratin material in the middle layer may be partially exposed by openings in the two outer nonwoven synthetic polymer layers.
Other aspects of the present invention include wound dressings, diapers and feminine hygiene products which comprise a nonwoven film made from a synthetic polymer and a hydratable keratin material. In certain embodiments, the non-woven film of the present invention may be next to the skin or other epithelial layer of a subject, or may be separated from the skin or other epithelial layer of a subject by a water permeable layer, which may be a non-wetting water permeable layer. In certain embodiments a hydratable keratin solid may be associated with a nonwoven layer of a diaper, or may be impregnated into a layer of a disposable diaper, or it may be contained in an inner absorbent core. These products may be laminate compositions, which may be tri-laminates comprising two outer layers of synthetic polymer and a middle layer of keratin material. The keratin material in the middle layer may be partially exposed by openings in the two outer nonwoven synthetic polymer layers. In some embodiments of the invention the synthetic polymer layers are nonwoven webs of polymer.
Creams, lotions, or gels of the present invention may incorporate or replace other ingredients known in the art, including, but not limited to oleaginous, emulsifiable, emulsion base, or water-soluble ointment bases as are well known in the pharmaceutical arts. Oleaginous bases that may be combined with the keratin compositions include ointments containing white wax and/or white petrolatum, ointments containing yellow wax and petrolatum, cetyl esters wax, oleic acids, and paraffins. Absorbent ointment bases or emulsifiable bases that may be used include those containing anhydrous lanolin, or combinations of cholesterol, stearyl alcohol, white wax and petrolatum, for example. Emulsion bases and components that may be used include ointments containing cetyl alcohol, and cold creams such as those containing cetyl esters wax, white wax, mineral oil, sodium borate and water, for example. Other ointments of the present invention may contain glyceryl monostearate, lanolin, stearic acid, or a combination of methylparaben, propylparaben, sodium lauryl sulfate, propylene glycol, stearyl alcohol and white petrolatum, for example, or an ointment containing cetyl esters wax, white wax, almond oil, sodium borate, stronger rose water, and rose oil, for example. Water soluble ointments and creams for use in the present invention may include glycol ethers and derivatives thereof, polyethylene glycols, polyoxyl 40 stearate, and/or polysorbates.
The preparations as described herein for topical applications may also include protectives and absorbents, demulcents such as benzoin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, propylene glycols, sodium alginates, and tragacanth. Emollients, astringents, or antiperspirants may also be included in the keratin containing formulations as described herein.
An aspect of the present disclosure is a method for augmenting soft tissue in a subject comprising injecting a keratin composition as described herein subdermally in an area in need of augmentation. A variety of such applications are available in light of the present disclosure and would include augmentation of soft tissue including, but not limited to bulking of a urinary sphincter in order to alleviate urinary incontinence, augmentation of vocal chords to restore elasticity, as well as improvement of the appearance of a subject by augmentation of breasts, lips, chin, gluteal area, or even to improve wrinkled or acne scarred skin, or skin scarred by other conditions, and including soft tissue voids or indentations. A keratin composition may be provided as a dry solid and hydrated after subdermal implantation, or a hydrogel or viscoelastic hydrogel may be prepared and implanted. In certain embodiments, a dry or hydrated keratin material may be contained in a biocompatible envelope, bag, or container for subdermal implantation, and hydrated after implantation by addition of water or absorption of body fluids, or a keratin material may be suspended in an injectable carrier and injected in the desired area of augmentation. In one embodiment, soft tissue augmentation is accomplished by injecting a preparation comprising a keratin hydrogel and a cell population.
Another aspect of the present invention is the use of keratin compositions for tissue engineering applications. One embodiment is an implantable preparation comprising a keratin hydrogel and a cell population. The cell population may include, but are not limited to, keratinocytes, fibroblasts, chondrocytes, hepatocytes, splenocytes, neurocytes, osteoblasts, or endothelial cells. The keratin hydrogel preparation may be prepared such that it is implantable by injection. Through processing and/or formulation parameters, the keratin hydrogel may be prepared such that is resorbable and that such resorption is at a controlled rate. The keratin hydrogel may be prepared such that it is resorbed upon implantation after about 150 days, or about 100 days, or about 90 days, or about 80 days, or about 70 days, or about 60 days, or about 50 days, or about 40 days, or about 30 days, or about 20 days, or about 10 days, or between about 10 and about 90 days, or between about 20 and about 90 days, or between about 50 and about 90 days, or greater than 90 days. The keratin hydrogel may also contain a therapeutic agent, which may be a water soluble peptide, which may be a mitogen.
Another embodiment is a cell scaffold comprising a nonwoven film which comprises a hydratable keratin which contains sulfonate groups. The nonwoven film may comprise a synthetic polymer which may be resorbable. The hydratable keratin may also be made resorbable, thereby rendering the entire nonwoven film resorbable. The nonwoven film may also be a laminate composition. The hydratable keratin may contain a therapeutic agent which may be a water soluble peptide, which may be a mitogen.
Another embodiment of the present invention is a method of implanting a preparation comprising a population of cells and a keratin hydrogel into an animal. The cell populations may include, but are not limited to, keratinocytes, fibroblasts, chondrocytes, hepatocytes, splenocytes, neurocytes, osteoblasts, or endothelial cells. The hydrogel may contain a therapeutic agent which may be a water soluble peptide, which may be a mitogen. A further embodiment is a method of implanting a cell scaffold comprising a nonwoven film that comprises a hydratable keratin which contains sulfonate groups. Cells may be grown on the nonwoven cell scaffold in vitro. Thereby, the nonwoven cell scaffold may be seeded with cells prior to implantation. Such cells include, but are not limited to, keratinocytes, fibroblasts, chondrocytes, hepatocytes, splenocytes, neurocytes, osteoblasts, or endothelial cells. The nonwoven cell scaffold may be used to repair damaged hard and soft tissues. Hard tissues include, but are not limited to, bone and cartilage. Soft tissues include, but are not limited to, skin, mucosa and muscle. Mucosal tissue includes, but is not limited to, gingival tissue, which may be damaged bone or damaged cartilage. Another embodiment comprises implanting a keratin hydrogel cell scaffold. A therapeutic agent may be included in the keratin component of all the cell scaffolds of the present invention. Such a therapeutic agent may leach out of the keratin and may be a water soluble peptide, which may be mitogenic.
On one aspect of the present invention, the keratin composition is a preparation comprising a cross-linked insoluble oxidized keratin excipient. The preparation may be in the form of a powder, tablet, film, capsule, lotion, cream, gel, solution, suspension, emulsion or aerosol. The preparation may be a cosmetic preparation or may be a pharmaceutical preparation. The preparation may further comprise one or more additives such as diluents, fillers, lubricants, stabilizers, binders and gelants.
It is an aspect of the present invention that a keratin composition as described herein, and in particular keratin obtained from human hair is also useful as an excipient for the delivery of an active agent. An embodiment of the invention may be described, therefore, as a composition comprising a keratin having oxidized cysteic groups and an active agent or as a cross-linked insoluble oxidized keratin excipient with an active agent. In certain embodiments the active agent is physically or sterically entrapped within the keratin excipient and released over time by diffusion, or as a keratin excipient is degraded. Further, in some embodiments the active agent may be associated with the keratin excipient. The association between the active agent and the keratin excipient may be by non-covalent attraction or association, through electrostatic, hydrophilic or ionic interaction, for example, or it may be covalently attached to a keratin excipient by covalent bonding to an oxidized keratin as described herein. In one embodiment, the active agent is in a cationic form that ionically binds to the sulfonate groups of the ionized keratin. In another embodiment the active agent is associated with the keratin excipient by Van der Waal""s forces. Association of the active agent with a keratin excipient allows for the sustained and/or controlled release of active agents. In some embodiments, the controlled release of the active agent is provided by the hydrolysis of the keratin excipient. Such a formulation may include a hydratable keratin solid excipient, or a keratin hydrogel depending on the particular application. In some embodiments the active agent is a pharmaceutical agent while in other embodiments the active agent is a cosmetic agent.
In the practice of the invention, a dry hydratable keratin as described herein may be mixed with a powdered pharmaceutical agent and water added to hydrate the mixture, or alternatively such a solid mixture may be formulated as a compressed tablet to be orally administered or for extemporaneous preparations for injection, or as a molded tablet, or it may be enclosed in a capsule for oral administration or subdermal implantation, for example. In certain embodiments a solution containing a water soluble drug or pharmaceutical agent may be added to a hydratable keratin so that the agent is carried into a hydrogel along with the water. A prepared hydrogel, or dry formulation may also be enclosed in a digestible or biodegradable capsule, such as a hard gelatin capsule for oral administration. In certain embodiments, the described pharmaceutical preparations may be formulated for injection, either intravenous, subcutaneous, or intramuscular, for example, or for inhalant, for eye, ear, or nose drops, or for administration as a suppository.
It is understood that the pills formulated for oral administration, including a hydratable keratin solid, or even pills, capsules or tablets containing a keratin hydrogel may contain ingredients to serve as coatings, additional fillers, binders and for color coding purposes. These ingredients are in common use in present pharmaceutical formulations and may include, but are not limited to, gelatin, lactose, corn starch, calcium phosphate, povidone, magnesium stearate, stearic acid, colloidal silicon dioxide, hydroxypropyl methylcellulose, polyethylene glycol and one or more of the following dyes: FDandC Blue No. I Lake, FDandC Blue No. 2 Aluminum Lake, DandC Green No. 5, DandC Yellow No. 10, FDandC Yellow No. 6 or FDandC Red No. 3. Of course these are only exemplary fillers and dyes, those of skill in the art will recognize that other inactive ingredients may be used in the preparation of the formulations of the present invention.
Keratin excipient preparations as described herein may be prepared for oral administration, and would also include injectable solutions or suspensions for intramuscular or subcutaneous implantation including long acting injections, suppositories, topical ointments, transdermal applications such as skin patches, and preparations delivered by inhalation. Other ingredients may include a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Keratin excipient preparations may also include other compounds such as diluents, fillers, lubricants, stabilizers, binders and gelants. Diluents and fillers are added to increase bulk formation, and lubricants to reduce friction during the tableting or other formulation process. Binders are used in tableting and provide the cohesiveness necessary for bonding together the ingredients under compression. They also increase the strength of the compressed tablet and decrease its friability, leading to an improvement in the both appearance and mechanical characteristics.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion, sterile powders and hydrogels for the extemporaneous preparation of sterile injectable solutions, dispersions or gels. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Suitable pharmaceutical agents for use with the excipients described herein would include any pharmaceutical agent that can form an association with the keratin formulations through non-covalent, covalent, or steric; interaction. These agents would include protein therapeutic agents, such as growth factors. In regard to oral administration, such agents may include compounds such as acetaminophen, tetracyclines, penicillins, vitamins, antacids, non-steroidal antiinflammatory agents, anesthetics, breath fresheners, and minerals, for example.
In those embodiments in which transdermal administration is desired, the disclosed compositions may be formulated to be administered by use of a skin patch, or transdermal delivery system. Transdermal administration may be accomplished by any of a number of systems known in the art. Examples of systems that may be adapted for use with the compositions described herein include those systems of transdermal administration described in U.S. Pat. No. 4,816,252; U.S. Pat. No. 5,122,382; U.S. Pat. No. 5,198,223; U.S. Pat. No. 5,023,084; U.S. Pat. No. 4,906,169; U.S. Pat. No. 5,145,682; U.S. Pat. No. 4,624,665; U.S. Pat. No. 4,687,481; U.S. Pat. No. 4,834,978; and U.S. Pat. No. 4,810,499 (all incorporated herein by reference).
These methods typically include an adhesive matrix or drug reservoir system and may include a skin permeation enhancement agent such as ethanol, polyethylene glycol 200 dilaurate, isopropyl myristate, glycerol trioleate, linolenic acid saturated ethanol, glycerol monooleate, glycerol monolaurate, n-decyl alcohol, capric acid, and certain saturated and unsaturated fatty acids, and their esters, alcohols, monoglycerides, acetate, diethanolamides and N,N-dimethylamides (See for examples, U.S. Pat. No. 4,906,169).
The release rate of an active agent from a keratin excipient preparation, when that active agent is not associated with the keratin excipient, is determined by the rate at which water is absorbed and the keratin solid disintegrates. The water absorption rate of the solid keratin can be controlled by the number of sulfonic acid residues generated in the oxidation procedure. By exposing the keratin source material to extremes of oxidant concentration, temperature, and time, extremes of absorption rate can be obtained. For example, at low oxidant concentration, colder temperatures and short time periods, relatively few disulfide residues will be converted to sulfonic acid residues. Such a keratin solid, further processed as described herein will absorb relatively little water and disintegrate relatively slowly. Conversely, a keratin solid prepared at high oxidant concentration, at boiling temperature for a long time period, further processed as described herein, will absorb relatively large amounts of water and disintegrate relatively quickly. Disintegration rates between these extremes can be obtained by processing the keratin source material using intermediate conditions. When the active agent is associated, such as ionically, with the keratin excipient, the release rate is determined by both the rate at which water is absorbed and the keratin solid disintegrates and the rate of dissassociation of the active agent from the keratin excipient. For some materials which are so tightly bound that release by dissassociation alone is ineffectual, degradation of the keratin solid must occur before the drug molecule can become dissolved in the surrounding media. The release rate under these conditions can be controlled by the degradation rate of the keratin solid. In general, oxidation and formulation conditions will effect the hydrolytic stability of the hydrogel containing the drug compound. Peracetic acid oxidized keratin provides a more hydrolytically stable gel than does hydrogen peroxide oxidized keratin, for example. Parameters such as oxidant, oxidation time and solids content of the hydrogel have been shown to be important parameters in controlling in-vitro stability at body temperature.
In some embodiments of the invention, a composition for the delivery of pharmaceutical agents is in the form of a nonwoven film comprising a synthetic polymer and a keratin material. The synthetic polymer may be, but is not limited to, xcex1-olefins, acrylates, urethanes, acetates, nylons, esters, and copolymers thereof. In some embodiments the nonwoven composition is a laminate, which may be a tri-laminate comprising two outer layers of synthetic polymer and a middle layer of keratin material. The keratin material in the middle layer may be partially exposed by openings in the two outer nonwoven synthetic polymer layers. In some embodiments of the invention the synthetic polymer layers are nonwoven synthetic polymer webs. The nonwoven film pharmaceutical delivery composition may be used externally or internally.